Perception of sound and balance depends on mechanotransduction, whereby a molecular complex at the tips of hair cell stereocilia converts an external mechanical stimulus into an electrical signal, which can propagate to the central nervous system. A key unknown in auditory neuroscience is the identity of the molecules that constitute the ion-conducting transduction channel within this complex. By studying deafness- linked genes in humans and mice, investigators have suggested that TMC1 and TMC2 are strong mechanotransduction channel candidates. Unfortunately, biochemical methods with sufficient sensitivity and selectivity to robustly detect and quantify the TMCs or other membrane proteins within the mechanotransduction complex have yet to be developed. Antibody-based methods such as Western blotting and immunocytochemistry often suffer from poor specificity, low multiplexing capabilities, and imprecise quantitation, thus preventing them from providing accurate quantitative information about the composition of the mechanotransduction complex. In addition, the high cost and limited availability of quality antibodies, especially for membrane proteins, limits screening for new transduction-channel candidates identified by other means. The goal of this proposal is to characterize the composition of the mechanotransduction complex in hair cells. To do so, we will develop targeted proteomic assays to identify and accurately quantify transduction proteins and measure how their stereocilia location and mechanotransduction complex association are altered in mouse models of deafness and vestibular dysfunction. We will design two sets of assays, which will allow us to simultaneously monitor the concentrations of known mechanotransduction complex proteins, as well as detect new candidate transduction channel proteins identified in hair cells at the RNA level. These assays, combined with other biochemical approaches, will allow us to 1) determine whether TMC1, TMC2, or other channel-like membrane proteins are localized to the hair bundle and are able to interact with other transduction complex proteins, and 2) determine how the localization of mechanotransduction proteins change in mice lacking TMC1 and TMC2. Together, the results of these studies will significantly contribute to our understanding of the molecular basis of hearing and deafness. In the long term, these assays will continue to be valuable tools for assessing how the composition of the mechanotransduction complex changes across development in various mouse models, as well as in the context of hair cell repair and regeneration.